Recognition signal for C-mannosylation of Trp-7 in RNase 2 consists of sequence Trp-x-x-Trp.
C2-alpha-Mannosyltryptophan was discovered in human RNase 2, an enzyme that occurs in eosinophils and is involved in host defense. It represents a novel way of attaching carbohydrate to a protein in addition to the well-known N- and O-glycosylations. The reaction is specific, as in RNase 2 Trp-7, but never Trp-10, which is modified. In this article, we address which structural features provide the specificity of the reaction. Expression of chimeras of RNase 2 and nonglycosylated RNase 4 and deletion mutants in HEK293 cells identified residues 1-13 to be sufficient for C-mannosylation. Site-directed mutagenesis revealed the sequence Trp-x-x-Trp, in which the first Trp becomes mannosylated, as the specificity determinant. The Trp residue at position +3 can be replaced by Phe, which reduces the efficiency of the reaction threefold. Interpretation of the data in the context of the three-dimensional structure of RNase 2 strongly suggests that the primary, rather than the tertiary, structure forms the determinant. The sequence motif occurs in 336 mammalian proteins currently present in protein databases. Two of these proteins were analyzed protein chemically, which showed partial C-glycosylation of recombinant human interleukin 12. The frequent occurrence of the protein recognition motif suggests that C-glycosides could be part of the structure of more proteins than assumed so far.
Krieg
J
J
Friedrich Miescher-Institut, CH-4002 Basel, Switzerland.
Hartmann
S
S
Vicentini
A
A
Gläsner
W
W
Hess
D
D
Hofsteenge
J
J
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002244:Carbon / Q000737:chemistry; D002460:Cell Line; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D007668:Kidney; D008364:Mannosyltransferases / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017510:Protein Folding; D011993:Recombinant Fusion Proteins; D013552:Swine; D014364:Tryptophan / Q000737:chemistry*
D011993:Recombinant Fusion Proteins; D002244:Carbon; D014364:Tryptophan; D008364:Mannosyltransferases; D004722:Endoribonucleases; C029494:ribonuclease U